Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos

Abstract

Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys.

Preliminary Molecular Studies of the First Report of Burkholderia pseudomallei Isolation from Soil Collected in the Amapá State, in Northern Brazil

“…Similarly to our findings of B. pseudomallei isolation from mangrove, it has been commonly reported the microorganism isolation from moist soil and water (Prakash et al, 2014;Knappik et al, 2015;Liu et al, 2015). Also, the high pluviometric index in the Amapá state (Segovia et al, 2011), as part of the Amazon region of Brazil, contributes to maintain the elevated soil humidity and natural collections of water.…”

Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples

“…Enrichment of soil specimens increases the concentration of B . pseudomallei in the broth exponentially, and the enrichment step (for 1–2 days) is already necessary for both culture and molecular detection [17,18]. We show that, for the evaluated LFI, enrichment of only 2 days is not sufficient, and 7 days are needed to increase the quantity of B .…”

“…pseudomallei . Second, qPCR has a lower detection limit compared to the culture method [18], and could also detect non-culturable B . pseudomallei [27].…”

“…The qPCR assay targeting orf2 of B . pseudomallei type III secretion system (TTS1) was performed as previously described [18]. …”

“…pseudomallei on agar plates among other soil microbes is time-consuming and requires expertise and experience. For effective molecular detection, soil specimens are also first enriched in selective broth for 2 days prior to nucleic acid amplification [17,18]. Despite the higher sensitivity than culture, PCR assays also require experienced personnel and positive controls, and are relatively costly for resource-limited settings.…”